Schaffner W, Gross K, Telford J, Birnstiel M
Cell. 1976 Aug;8(4):471-8. doi: 10.1016/0092-8674(76)90214-2.
Histone DNA of Psammechinus miliaris was obtained in an enriched form by buoyant density gradient centrifugation and was cleaved into 6 kb repeat units (Birnstiel et al., 1975a) by the action of the specific endonucleases EcoRI and HindIII. Since it was suspected that the 6 kb unit harbored all five histone-coding sequences, the histone DNA unit was subdivided into five segments with the aim of providing five fragments carrying just one coding sequence each. This was achieved by the combined use of EcoRI Hindll, Hindlll, and Hpa I. A physical map was constructed from the overlaps arising in these restriction experiments. Each of the five segments was shown to hybridize uniquely with just one of the five highly purified histone mRNAs (Gross et al., 1976a). By this procedure, the order of the mRNA sequences on the histone DNA was found to be a, c, d, b, e (Gross et al., 1976a), and hence of the protein coding sequences H4, H2B, H3, H2A, and H1. Further evidence is presented that the 6 kb repeat unit, amplified by means of a Murray lambda vector phage, contains AT-rich DNA sequences which would be expected not to code for histone proteins.
通过浮力密度梯度离心法获得了富集形式的沙钱组蛋白DNA，并通过特异性内切酶EcoRI和HindIII的作用将其切割成6 kb的重复单元（伯恩斯蒂尔等人，1975a）。由于怀疑6 kb的单元包含所有五个组蛋白编码序列，因此将组蛋白DNA单元细分为五个片段，目的是提供五个分别仅携带一个编码序列的片段。这是通过联合使用EcoRI Hindll、Hindlll和Hpa I实现的。根据这些限制性实验中出现的重叠构建了物理图谱。五个片段中的每一个都被证明仅与五个高度纯化的组蛋白mRNA之一特异性杂交（格罗斯等人，1976a）。通过这个过程，发现组蛋白DNA上mRNA序列的顺序是a、c、d、b、e（格罗斯等人，1976a），因此蛋白质编码序列是H4、H2B、H3、H2A和H1。进一步的证据表明，通过默里λ载体噬菌体扩增的6 kb重复单元包含富含AT的DNA序列，预计这些序列不编码组蛋白。
